In vitro transcription systems are used to identify promoter signals and factors that control transcription in eucaryotic sytems. These systems accurately initiate transcription from a wide variety of promoters. Preferential stimulation of certain promoters is achieved in the presence of low concentrations of ammonium sulfate. The addition of (NH4)2S04 allows detection of promoters which could not otherwise be detected and enhances in vitro expression of the late promoter 5' start site that is used as the principal in vivo start site. Under these modified conditions, expression of the adenovirus IVa2 gene is readily observed in vitro, while under standard conditions, it is not transcribed. This adeno gene, like the SV40 late genes, lacks a TATA box upstream from the 5' start site. By constructing a series of upstream deletion mutations, two upstream domains have been identified that controls this gene. The distal domain only functions when covalently closed DNA is used as a template.